Assembly intermediates of the mouse muscle nicotinic acetylcholine receptor in stably transfected fibroblasts.
We have used fibroblast clones expressing muscle nicotinic acetylcholine receptor alpha and gamma, and alpha and delta subunits to measure the kinetics of subunit assembly, and to study the properties of the partially assembled products that are formed. We demonstrate by coimmunoprecipitation that assembly intermediates in fibroblasts coexpressing alpha and delta subunits are formed in a time-dependent manner. The alpha and gamma- and the alpha and delta-producing transfected cells form complexes that, when labeled with 125I-alpha-bungarotoxin, migrate in sucrose gradients at 6.3S, a value consistent with a hetero-dimer structure. An additional peak at 8.5S is formed from the alpha and gamma subunits expressed in fibroblasts suggesting that gamma may have more than one binding site for alpha subunit. The stability and specificity of formation of these partially assembled complexes suggests that they are normal intermediates in the assembly of acetylcholine receptor. Comparison of the binding of 125I-alpha-bungarotoxin to intact and detergent-extracted fibroblasts indicate that essentially all of the binding sites are retained in an intracellular pool. The fibroblast delta subunit has the electrophoretic mobility in SDS-PAGE of a precursor that does not contain complex carbohydrates. In addition, alpha gamma and alpha delta complexes had lectin binding properties expected of subunits lacking complex oligosaccharides. Therefore, fibroblasts coexpressing alpha and gamma or alpha and delta subunits produce discrete assembly intermediates that are retained in an intracellular compartment and are not processed by Golgi enzymes.
Blount
P
P
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Smith
M M
MM
Merlie
J P
JP
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D002038:Bungarotoxins; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic